The capacity of the immune system to prevent the expansion of autoreactive B and T cells is a critical step in the prevention of autoimmunity, and T cell anergy may play a role in maintenance of peripheral tolerance. Although there exist many molecules that provide costimulatory signals to T cells, only one signal, that mediated by the T cell surface protein CD28, has been shown to prevent the induction of T cell anergy. Manipulation of costimulatory pathways may provide new approaches to treating autoimmune disease and cancer; however, little is known about the costimulatory requirements of CD28" T cells which account for up to half of human CD8+, CD4"8" alphabetaTCR+, and gammadeltaTCR+T cells. In order to understand the pathways of activation used by CD28" T cells, we have examined the costimulatory requirements of antigen specific human CD4"CD8" alphabetaTCR+ T cell lines that lack the expression of CD28. Although the T cells recognized antigen presented by both CD1+ monocytes and CD1+ B lymphoblastoid cells (B-LCL), proliferation occurred only when antigen was presented by the CD1+ monocytes. Furthermore, when the T cells were cultured with antigen pulsed CD1+ B-LCL, T cell anergy was induced. This suggested that the CD1+ monocytes expressed a costimulatory molecule that the B- LCL transfectants lacked. The required signal occurs via a CD28- independent mechanism and represents a previously unrecognized pathway of costimulation for T cells. This grant proposes to use human T cell lines that require a non-CD28 costimulatory signal to identify the proteins involved in this novel monocyte dependent costimulatory pathway. In Aim 1, the costimulatory ligand expressed by CD1+ monocytes will be identified and characterized. Known costimulatory molecules will be examined for their ability to prevent anergy in DN1 T cells. If a known molecule is not identified as the relevant costimulatory molecule, antibo will be generated and screened functionally in order to identify the relevant molecule. Alternately, a method to directly clone the gene encoding the costimulatory molecule will be developed. Finally, the cDNA encoding the costimulatory molecule will be transfected into the nonstimulatory CD1+ B-LCL to determine if it confers the ability to stimulate the proliferation of DN1 T cells. In Aim II, antigen specific MHC restricted T cell clones that are CD28- will be derived and used to further study this novel costimulatory pathway and ascertain the relevance of this pathway to MHC restricted T cells.